To genetically perturb the function of the LRRTM4-HSPG complex in mice in vivo, we focused on LRRTM4 because multiple proteoglycans can interact with LRRTM4 and we expect that

deletion of multiple proteoglycans would be required to perturb the function of LRRTM4-HSPG signaling. We generated mice with a targeted deletion in LRRTM4 by deleting exon 2, which encodes a large portion of the LRRTM4 protein ( Figures S4A). Loss of LRRTM4 protein was confirmed by western blot analysis of whole mouse brain homogenate ( Figure 6A) and by confocal microscopy selleck inhibitor analysis of brain sections with an anti-LRRTM4 antibody ( Figure 6B). LRRTM4−/− mice were viable and fertile and indistinguishable from wild-type mice with respect to gross brain morphology and cytoarchitectural organization as assessed by confocal microscopy analysis of brain sections labeled for the nuclear marker DAPI, the synaptic marker synapsin, the dendritic marker MAP2, and the axonal marker dephospho-tau ( Figures 6B

and 6C and data Selleckchem HIF inhibitor not shown). Given the high levels of LRRTM4 in the molecular layers of dentate gyrus, we tested whether levels of HSPGs and key postsynaptic molecules may be altered in the dentate gyrus of LRRTM4−/− mice. We prepared crude synaptosomal fractions from isolated dentate gyri from LRRTM4−/− and control wild-type mice at 6–7 weeks postnatally, a time when LRRTM4 expression reaches a plateau ( Figure 1A). Quantitative immunoblotting of these fractions revealed no difference between LRRTM4−/− and wild-type mice in the level of AMPA receptor subunits GluA1 and GluA2 ( Figures 6D and 6E). While the level of the inhibitory synapse scaffolding molecule gephyrin remained unchanged, the level of PSD-95 no family proteins was significantly reduced in LRRTM4−/− mice, indicating that LRRTM4 is an important component of excitatory postsynapses in the dentate gyrus. Next, we determined whether the level of HSPGs may be affected by the loss of LRRTM4. Representatives of glypicans and syndecans, GPC2 and SDC4 were both significantly reduced in

the crude synaptosomal fractions of LRRTM4−/− mice dentate gyri. Moreover, using an antibody that recognizes the glycosaminoglycan stub region after heparinase treatment, we found that the total level of all HSPGs in crude synaptosomal fractions of LRRTM4−/− mice dentate gyri was significantly reduced, indicating that LRRTM4 is an important functional partner of HSPGs. We next performed confocal imaging of excitatory and inhibitory synaptic markers in LRRTM4−/− dentate gyrus molecular layers, in comparison with CA1 stratum oriens, a region where LRRTM4 is not expressed ( Figures 1 and 6; Laurén et al., 2003 and Lein et al., 2007), again at 6–7 weeks postnatally. Quantitative confocal analysis revealed reduced punctate VGlut1 immunofluorescence in all dentate gyrus molecular layer regions but not in CA1 stratum oriens in LRRTM4−/− mice as compared with wild-type littermates ( Figures 6F and 6G).